Electrofusion of brewers' yeast protoplasts and enrichment of the fusants using a flow cytometer

Electrofusion of brewers’ yeast protoplasts and enrichment of the fusants using a flow cytometer Naoto Urano, Brewing

Research

Hirohisa Sahara and Shohei Koshino Laboratories,

Sapporo

Breweries

Ltd.,

Shizuoka,

Japan

A sorting system of fusants between prototroph industrial yeasts by electrofusion was constructed usingflow cytometry. We previously reported conversion of a nonjlocculent brewers’ yeast to flocculent by electrofusion. The fusants contained undesired phenotypes from S. cerevisiae for brewing. In this study, refusion between the fusant and the brewers’ yeast was carried out and refusants were enriched using a flow cytometer. One-parent protoplasts were labeled with concanavalin A lectin-conjugated Jluorescein isothiocyanate, and both parent protoplasts were mixed and fused by electrofusion. The ef$ciencies of the refusants in all regenerated yeasts from protoplasts were 4-7 per 1,000. Labeled protoplasts after electrofusion were sorted using the flow cytometer and regenerated; the efficiencies of the refusants were 82-134 per 1,000. Therefore, the refusants were enriched 12-34 fold using the flow cytometer and sorting of them from regenerated yeasts was attained.

Keywords:

Electrofusion;

flow cytometer;

brewers’ yeast; fusant; Saccharomyces

Introduction In 1977 Solingen and Plaat’ reported protoplast fusion of Saccharomyces cerevisiae in the presence of polyethylene glycol. Since then, protoplast fusion has become a very important tool in the hybridization of yeasts. In the brewing field, protoplast fusion is also considered to have much potential for breeding of brewers’ yeasts. More recently, electrofusion has been widely utilized instead of the polyethylene glycol method because of its simplicity and high fusion efficiency. However, breeding of yeasts by these techniques has two common problems: First, separation of fusants from all regenerated yeasts is very difficult because industrial yeasts are generally prototroph and lack suitable selection markers for the fusion. Second, undesired phenotypes are often introduced into the yeasts as well as desired phenotypes, according to the fusion. In order to solve the first problem, we have studied the optimization of electrofusion condition using Shimadzu somatic hybridizer SSH-1 including some physical parameters2 and developed a novel cell fusion chamber named fluid integrated circuit.3 How-

cerevisiae

ever, fusants between brewers’ yeasts could not be constantly separated by these devices because protoplasts of the yeasts have very low efficiency for both regeneration and fusion. Therefore, a new enrichment system of the fusants was needed. In recent years, flow cytometry has been used for analysis of various kinds of parameters or materials in protoplast fusants such as plasmid stability,4 cell cycles,5,6 viability,’ ploidy level,8-” and binding of bacteria and plant cells.‘* However, no studies have been reported about sorting of yeast protoplasts using flow cytometry. In this study, a flow cytometer was used for the enrichment of the fusants between brewers’ yeast protoplasts by electrofusion. In the previous studies we reported conversion of a weak or a nonflocculent brewers’ yeast to strong flocculent yeast by electrofusion between a brewers’ yeast and S. cereuisiae B2-1 (FL05).‘3-15 However, the fusants contained undesired phenotypes from 5. cereuisiae for brewing. Therefore, refusion between one of the fusants and a brewers’ yeast, and screening of the refusants which contain only desired phenotypes were carried out in the present study in order to solve the second problem. Materials and methods

Address reprint requests to Dr. Urano at the Brewing Research Laboratories, Sapporo Breweries Ltd., Yaizu, Shizuoka 425, Japan Received 25 November 1992; revised 18 March 1993

0 1993 Buttetworth-Heinemann

Yeast strains Yeast

strains

used are listed

Enzyme Microb. Technol.,

in Table 1.

1993, vol. 15, November

959

Papers Table 1

Yeast strains

used in this study

[iI-ykzq

Strain

Source

Saccharom yces cerevisiae G706 (a his& leu2, thr4) G706-1 (a his4, leu2, thr4, p-1 0708-l I-8A (a ura 1, adel) 0708-ll-16A (a ura7, adel) ABXR-11 B (a FLO5) 82-l

(a ura7, adel,

Brewers’ yeast BSRI YB9-12 BSRI YB9-12-1 F(80) (FL051

1305)

Brewer’s

Prof. N. Gunge (Kumamoto Institute of Technology, Japan)

yeast

BSRI

12. YB9-12-1

v

Fusant ‘L-r’ with

Treatment

cerevisiae

F(80)

Zymolyase

20T

Protoplasts I

Yeast Genetic Stock Center Mating 0708-I l-8A with ABXR-11 B Breweries

.

1

.

Protopklsts

Sapporo

0708-ll-16A

Filtered

through

Net

(53

urn)

i Recovered

Protoplasts

Ltd.

AC

(0-1

(1

MHZ,

for

Fusing BSRI YB9-12-1 with B2-1

400

10

s)

K”

cm

v

cm

-1 ,

v Pearl-chain

of

Protoplasts

DC

(4.5

-1 , 25 ps,

5 charqel

Preparation

*

of the yeast protoplasts

Membrane-fused

cells were grown aerobically overnight in YEPD medium [glucose (2%); peptone (2%); yeast extract (l%)] at 28°C. The cells were harvested and subjected to protoplasting with zymolyase 20T (200-400 pg 10m9 cells at 30°C for 120 min) in KC1 (0.4 M), Tris-HCl (10 mM) buffer at pH 7.5 containing bovine albumin (0.1%). Protoplasts were collected by centrifugation and resuspended in sorbitol(0.7 M). The samples were filtered through stainless nets (pore size 53 pm), both the cell wall debris and aggregated protoplasts were removed, and the filtrate was recovered as the protoplast preparation.

Protoplasts

Yeast

Sorting

by

a

flow

cytometer

1 Protoplasts

with

Fluorescence

Spread and

into

medium

R

incubated

at

28°C.

and flow

cytometry

+ Regenerated

Figure 1

Procedure

yeasts

for electrofusion

Labeling of protoplasts with concanavalin A lectin-conjugated fluorescein isothiocynate Concanavalin A lectin-conjugated fluorescein isothiocynate (Con A-FITC, EY Labs, Inc.) (0.5 mg) was added to S. cereuisiae G706-1 or brewers’ yeast BSRI YB9-12-l protoplasts (log), and the suspension was gently shaken at 0°C for 2 h. Labeled protoplasts were collected by centrifugation, the Con A-FITC remaining in solution was removed, and the sample was resuspended in sorbitol (0.7 M).

Electrofusion

of protoplasts

Two protoplast fusion systems were used in this study. The first was the fusion between S. cerevisiae G706-1 and S. cerevisiae 0708-ll-16A, and the second was that between brewers’ yeast BSRI YB 9-12-1 and the fusant, F(80). Parent protoplasts were mixed in a ratio of 1 : 1, and their total concentration was adjusted to 10’ cells ml-‘. Electrofusion was carried out using a Shimadzu somatic hybridizer SSH-1 (Shimadzu Co.) under optimum conditions according to Urano et a1.4 The procedure is shown in Figure 1. This process was observed by a reflected-light fluorescence microscope IMT2-RFL, a CCD high-gain color camera HCC-1600A, a still video recorder SR-300 (Olympus Optical Co.), and a color video printer UP-5000 (Sony Co. Ltd.).

Sorting of fusants or refusants using a flow cytometer (FMC) From all protoplasts treated with electrofusion (lo5 ml-‘), labeled cells were continuously sorted on an FMC (Epics 751, Coulter Electronics, Inc., Hialeah, FL) with 270 mV, 488 nm argon laser excitation using a 525 nm (* 25 nm) band

960

Enzyme Microb. Technol.,

pass filter for FITC. The sheath solution used was 2.8 PBS( -) buffer (Nissui Pharma. Co. Ltd.).

Regeneration of protoplasts fusants or refusants

x

and screening oj

Both before and after sorting with an FMC, protoplasts were regenerated in medium A [glucose (0.1%); glycerol (2%); yeast extract (2%); Bacto peptone (1%); yeast nitrogen base (0.67%); sorbitol (0.3 M); agar (1.5%)] at 28°C for 1 week. In fusion system I, regenerated yeasts were replicated to the minimum medium [glucose (2%); yeast nitrogen base (0.67%); agar (2%)] and colonies grown were considered to be fusants. In fusion system II, flocculation activities of regenerated yeasts were assayed by the method of Johnston and Reader.‘” After cultivation in YEPD medium (10 ml) at 15°C for 1 week, the sedimented yeasts were suspended in sodium acetate (0.05 M, pH 4.5), CaCl, (5 mM) solution (10 ml). The suspension was transferred to a test tube (30 ml) and stirred by a mixer. After standing at room temperature for 30 s, flocculation activity was visually estimated and expressed on a subjective scale ranging from 0 (nonflocculent) to 5 (strongly flocculent). A brewers’ yeast BSRI YB9-12-l was in 0 grade and a fusant F(80) was in 4 grade. The yeasts in grades l-3 were considered to be refusants between them.

Analysis of chromosomal DNA of fusants or refusants by pulsed field gel electrophoresis DNA samples from the yeast strains were prepared by the modified method of Carle and Olson.” Protoplasts were pre-

1993, vol. 15, November

Electrofusion pared as mentioned above; 5 g of protoplasts were suspended in sorbitol (0.7 M) and mixed with 7.5 ml of agarose (1%) (SeaKem GTG, FMC BioProducts, USA) at 50°C. They were poured into proteinase K (1%) (Boehringer Mannheim GmbH, Germany), EDTA (0.5 M), Tris-HCI (50 mM), sarkosyl (1%) (pH 9.5) at 50°C for 2 days. Analysis of chromosomal DNA in each sample was carried out by electrophoresis unit AE-6800 (Atto Co. Ltd.). The electrophoretic conditions employed were agarose (1.5%), 180 V at 12°C for 44 h and a switching interval of 25-50 s.

of brewers’

yeast protoplasts:

N. Urano

et al.

10 flrn -

Results and discussion Observation

of electrofusion

Figure 2A-C

shows photographs of successive steps in electrofusion of parent protoplasts under fluorescent light at 334-365 nm. Both parent protoplasts in fusion system II were mixed 1 : 1 and exposed to an alternating electric field (1 MHz, 400 V cm- I). Pearl chains consisting of several protoplasts were formed, and only one parent brewers’ yeast BSRI YB9-12-l with fluorescence was observed under the microscope, as shown in Figure 2A. When pulse (5 kV cm, 25 ps x

5) was charged to the chains, several multimembranefused protoplasts appeared among them, as shown in Figure 2B. They were in long states a few seconds after charging pulse and gradually shortened. After 30-60 min the protoplasts became spherelike, as shown in Figure 2C. The membrane-fused protoplasts with various hybrid ratios of both parents were observed to be constructed. The cells of 12-30 pm were thought to be membrane-fused protoplasts, and those of 5-10 pm were thought to be unfused brewers’ yeast. Under visible light, approximately the same number of cells without fluorescence were observed as those with fluorescence (data not shown). Using a flow cytometer, the protoplasts with fluorescence were sorted in this study. Sorting of Con A-FITC-labeled using a flow cytometer

protoplasts

Figure 3A shows flow cytometric observations of cell distributions in electrofused protoplasts in fusion system II. Peak I was a mass of Con A-FITC-labeled protoplasts, and peak 2 was intact protoplasts. Continuous sorting of each kind of protoplast was carried out using a flow cytometer. Two isolates were separately detected using the flow cytometer, and the purification efficiency of them was investigated. Figure 3B shows flow cytometric observations of labeled isolate. As the histogram contained peak 1 and a slight amount of peak 2, the isolate was considered to be mostly labeled. Figure 3C shows flow cytometric observation of intact isolate. As the histogram contained peak 2 and a slight amount of peak 1, the isolate was considered to be mostly intact protoplasts. The former isolate was spread into medium A and regenerated at 28°C for 1 week. Effect offlow of protoplasts

cytometry

on regeneration

As an argon laser irradiates intact or Con A-FITClabeled protoplasts using a flow cytometer, the effect of

Figure 2 Successive steps of electrofusion: (A) pearl-chain formation of protoplasts in an alternative field; (B) membrane-fused protoplasts in long states a few seconds after charging pulse; (C) membrane-fused protoplasts in spherelike forms 60 min after charging pulse

the laser on their regeneration was investigated. Both before and after sorting, protoplasts were spread into medium A, and the regeneration efficiencies in each kind of protoplast were compared, as shown in Table 2. In the case of intact protoplasts, the change of regeneration efficiency was 87-109% and was found to have little effect through the flow cytometer. On the other

Enzyme Microb. Technol.,

1993, vol. 15, November

961

Table 3

Ratio of fusants or refusants to all regenerated

yeasts

Condition

Ratio of fusants or refusants (x 10e3)

Before sorting After sorting Before sorting After sorting

4, 5, 9 56,72, 125 4, 6, 7 82, 106, 134

Fusion system (A) I II Ll._

0

50

100

Relative

fluorescence

150

Relative

200

c

250

intensity

100

50

L

Ratio of fusants is defined as follows: number of colonies growing in the minimum medium per 1,000 regenerated yeasts in medium A Ratio of refusants is defined as follows: number of yeasts with l-3 grades of flocculation activities per 1,000 regenerated yeasts in medium A

150

fluorescence

200

hand, in the case of Con A-FITC-labeled protoplasts, the change in efficiency was 23-47% through the flow cytometer. This phenomenon seems to be caused by the following steps: An exothermic reaction between FITC on the surface of labeled protoplasts and the laser occurred. The membrane lipid bilayer of the protoplasts became disordered a little. The protoplasts became weak against osmotic change, frequently burst and were spread into the regeneration medium, and caused the decrease in regeneration efficiency.

250

intensity

(C)

Regeneration &ants

0

50

100

Relative

150

fluorescence

200

250

intensity

Figure 3 Flow cytometric observation of protoplast distributions: (A) protoplasts after electrofusion, (B) the isolate of peak 1, (C) the isolate of peak 2. Peak 1: ConA-FITC-labeled protoplasts. Peak 2: nonlabeled protoplasts

Table 2

Effect of flow cytometry

on regeneration

of protoplasts

Regeneration efficiency (x 10-4)

Strain S. cerevisiae 0708-l l-l 6A S. cerevisiae G706-1 S. cerevisiae G706-1 (ConA-FITC-labeled) Fusant Ff80) Brewers’ yeast BSRI YB-9-12-1 Brewers’ yeast BSRI YB9-12-1 (ConA-FITC-labeled)

Before sorting

After sorting

After/before (%I

31 511 260

27 498 79

87 97 23

84 135 77

75 143 36

89 107 47

Regeneration efficiency is defined as follows: number of colonies growing in medium A per 10,000 protoplasts spread

962

Enzyme

Microb.

Technol.,

1993, vol.

15, November

of protoplasts

and screening

of

or refusants

Table 3 shows the ratio of fusants or refusants to all regenerated yeasts in both fusion systems. The ratio of the fusants before sorting was 4-9 per 1,000 regenerated yeasts, and that after sorting was 56-125 per 1,000 in fusion system I. Therefore, the enrichment of the fusant was 6- to 3 l-fold through a flow cytometer. The ratio of the refusants before sorting was 4-7 per 1,000, and that after sorting was 82- 134 per 1,000 in fusion system II. Therefore, enrichment of the refusants was 12- to 34-fold through the flow cytometer. The flow cytometer was found to be effective for sorting the fusants between prototroph yeasts. Identification of the fusants or refusants pulsed field gel electrophoresis

by

In order to ascertain the fusants from chromosomal DNA level, eight of the fusants screened in system I were compared to the parents by pulsed field gel electrophoresis, as shown in Figure 4. Both S. cereuisiae G706-1 and 0708-l l-16A showed the patterns of haploid cells. On the other hand, fusants a-h showed the patterns of diploid cells. For instance, the parents had chromosome (I), (VI), and (III) with different molecular weights, and all fusants had two bands of chromosome (I), (VI), and (III) from both parents. Therefore, the strains screened in the minimum medium were clearly identified as the fusants between both S. cereuisiae. In system II, eight of the refusants screened were compared to the parents, as shown in Figure 5.

Electrofusion 1,

2.

3.

4.

5.

.6.

7.

9,

8.

10.

(Ill,-

-(Ill) (VI)--(VI)

(I)--

-(I)

Figure 4 Electrophoretic patterns of chromosomes from parents and fusants in fusion system I. (1) fusant (a); (2) fusant (b); (3) fusant (c); (4) fusant (d); (5) S. cerevisiae G706-1; (6) S. cerevisiae 0708-II-16A; (7) fusant (e); (8) fusant (f); (9) fusant (9); (IO) fusant (h)

1.

2.

3.

4.

5.

6.

7.

8.

9,

10.

of brewers’

yeast protoplasts:

N. Urano et al.

in chomosome I of S. cereuisiae and the refusants might contain chromosome I from F(80). Protoplast fusion has been used to breed various kinds of auxotroph industrial yeasts such as yeast with killer character,19,20 yeast for ethanol production at high temperature,2’ yeast for lactose fermentation,22 yeast for starch fermentation23 using polyethylene glycol, and yeast for baking24 by electrofusion. Above all, as electrofusion appeared to offer some advantages over chemical methods, more detailed mechanisms and optimum conditions of the procedure were studied in detai1.2v25-28 However, there were few reports about practical applications of protoplast fusion to breeding of prototroph industrial yeasts. In the previous study, the fusant F(80) was in grade 4 of flocculation activity and had fairly undesired phenotypes from S. cerevisiae.‘4 Therefore, refusion between the brewers’ yeast and F(80) was carried out, and the refusants were enriched by the flow cytometer. From the point of flocculation of RF yeasts, the refusion seemed to be effective, and the availability of flow cytometry to sorting of fusants between prototroph yeasts could be demonstrated. Further studies are being directed towards more detailed characterization of the refusants obtained and application to practical brewing. Acknowledgement We are grateful to Dr. Masanobu Munekata, Dr. Akito Nakamura, and Mr. Toshiyuki Hashimoto, Pharmaceutical Research Laboratories, Sapporo Breweries Ltd., for providing the flow cytometric system and kind and useful suggestions. References Solingen, P. and Plaat, I. B. Fusion of yeast spheroplasts. Bacterial. 1977, 130, 946-947

Figure 5 Electrophoretic patterns of chromosomes from parents and refusants in fusion system Il. (1) RF9; (2) RF22; (3) RF89; (4) RF108; (5) brewers’ yeast BSRI YB9-12-1; (6) F(80); (7) RF175; (8) RF190; (9) RF195; (10) RF212

J.

Urano, N., Kamimura, M. and Washizu, M. Physical parameters affecting electrofusion of yeasts: Zeta-potential on the surface of yeast protoplasts and osmotic pressure of the solution. J. BiotechnoI. 1991, 18, 213-224 Urano, N., Kamimura, M., Nanba, T., Okada, M., Fujimoto, M. and Washizu, M. Construction of a yeast cell fusion system using a fluid integrated circuit. J. Biotechnol. 1991,20, 109-l 16 Drebot, M. A., Barnes, C. A., Singer, R. A. and Johnston, G. C. Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae. J. Bacterial. 1990, 172, 3584-3589

A brewers’ yeast BSRI YB9-12-1 had three bands of (I) and one band of (III). The fusant F(80) had one band of chromosome (I) and two bands of (III). On the other hand, RF9, RF22, and RF212 had three bands of chromosome (I) and two bands of (III), and were clearly identified as the refusants between the yeast BSRI YB9-12-1 and the fusant F(80). RF89, RF108, and RF175 had two bands of (I) and had a little different banding pattern from both parent strains. Both RF190 and RF195 had the same DNA patterns as the yeast BSRI YB9-12-1 in this condition. However, all the RF strains had l-3 grades of flocculation activities and were considered to have the FL05 gene from F(80). Vezinhet et al.” reported mapping of the FL05 gene

Dien, B. S. and Srienc, F. Bromodeoxyridine labelling and flow cytometric identification of replicating Succharomyces cereuisiae cells: Lengths of cell cycle phases and population variability at specific cell cycle posit&s. Biotechno?. Prog. 1991, 7, 291-298 Munch, T., Sonnleiter, B. and Fiechter, A. The decisive role of the Saccharomyces cerevisiae cell cycle behaviour for dynamic characterization. J. Biotechnol. 1992, 22, 329-352 Klock, G. and Zimmermann, U. Facilitated electrofusion of vacuolated x evacuolated oat mesophyll protoplasts in hypoosmolar media after alignment with an alternating field of modulated strength. Biochim. Biophys. Acta 1990, 1025, 87-93 Fahleson, J., Rahlen, L. and Glimelius, K. Analysis of plants regenerated from protoplast fusions between Brass& napus and Eruca saliva. Theor. Appl. Genet. 1988, 76, 507-512 Chaput, M. H., Sihachakr, D., Ducrex, G., Marie, D. and Barghi, N. Somatic hybrid plants produced by electrofusion

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Broda, H. Cl., Schnettler, R. and Zimmermann, U. Parameters controlling yeast hybrid in electrofusion: The relevance of preincubation and the skewness of the size distributions of both fusion partners. B&him. Biophys. Acta 1987, 899, 25-34 Emeis, C. C. Intergenetic hybridization of yeasts by electrofusion. 1Ith Jena Symp. Biophys. Chem.: Bioelectrochem. Biotechnol. (East Germany), 1987, pp. 31-34 Foerster, E. and Emeis, C. C. Enhanced frequency of karyogamy in electrofusion of yeast protoplasts by preceding G-l arrest. FEMS Microbial. Left. 1986, 34, 69-72 Schnettler, R., Zimmermann, U. and Emeis, C. C. Large-scale production of yeast Saccharomyces cerevisiae hybrids by electrofusion. FEMS Microbial. Left. 1984, 24, 81-86